Conventionally, a so-called protein complex, in which a protein is encapsulated in another protein, has been known. As for production of this type of protein complex, for example, a method of applying a solution of a dissolved protein to a surface of a crystalline protein is considered.
However, it is extremely difficult to carry out this method without dissolving the crystalline protein. Accordingly, the fact is that this method is hardly adopted for the purpose of protecting a useful protein (hereinafter referred to as a target protein) such as an enzyme, an antigen, an antibody, a cytokine or a receptor.
As for protection of a target protein, a method of covalently binding a polymer such as a polysaccharide polymer or polyethylene glycol to a target protein has been adopted. This method is a method in which a polymer is bound to a functional group such as an amino group or a carbonyl group in the target protein under mild reaction conditions. However, in this method, the binding site, the catalyzed site or the like of the target protein could not be controlled. In addition, since the binding site, the catalyzed site or the like varies depending on the type of the target protein, the method could not be applied to all the target proteins.
As for preservation of a target protein, generally, a method of preservation at a lower temperature is employed. In addition, a method of adding or mixing a protective substance (e.g., a polysaccharide polymer, polyethylene glycol and the like), which is expected to have a function of stabilizing the protein structure, to or with a target protein is also employed. However, by employing these methods, the stability or the function of the target protein was lost in some cases due to the changes in the environment, which is an external factor. That is, it is because the target protein is easy to dissolve together with the protective substance when water comes in contact, temperature or humidity increases, or dew condensation occurs. In addition, the target protein is degraded or ingested together with the protective substance when putrefactive bacteria such as germs or fungi exist, penetrate, or emerge. Therefore, when the target protein is a polymeric protein such as a protein molecule of some enzymes or antibodies, it lose its function completely by subjecting to a change in even a part of its structure or by degrading a part thereof with the action of a protease. However, when the target protein is used, it is essential that it sufficiently have its function. Therefore, it is necessary to verify the stability of the target proteins in a state of preservation individually. In the case of employing a conventional technique, it is necessary to take the target protein out of the protective substance, therefore, not only it takes a lot of time and efforts, but also the target protein is susceptible to denaturation.
By the way, cytoplasmic polyhedrosis virus forms a polyhedron composed of a polyhedral protein in a cell infected with the virus during the late phase of the viral infection, and many virus particles are embedded in the polyhedron.
The reason why the virus particles enter specifically in this polyhedron is known and it is due to the specific relationship between a capsid protein VP3 of the virus particle and a polyhedral protein (Non-Patent Document 1).
In view of the above-mentioned background, the present inventor completed the invention, which relates to a protein complex contributing to protection, preservation and improvement in stability of a target protein and a process for producing the same, and applied for a patent previously (Patent Document 1). The object of the description of the above-mentioned invention is to embed a polymeric target protein in this polyhedron and to enhance the embedding efficiency. Therefore, by shortening a gene encoding a capsid protein of cytoplasmic polyhedrosis virus, the size (molecular weight) of a protein which can be embedded in a polyhedron is made large, and this target protein is further more efficiently embedded in the polyhedron. Further, as the method, the amino acid sequence of VP3, which is a constituent protein of the envelope of cytoplasmic polyhedrosis virus, is introduced to the N-terminus or the C-terminus of the target protein, and this fusion protein is expressed with a baculovirus vector. At this time, by infecting an insect cell together with a virus expressing a polyhedral protein of cytoplasmic polyhedrosis virus, the fusion protein is embedded in a polyhedron. Accordingly, it is necessary to fuse a cDNA encoding a constituent protein of cytoplasmic polyhedrosis virus and a gene encoding a target protein so that a foreign protein expressed with a baculovirus vector, namely, a target protein is inserted at the N-terminus or the C-terminus of the constituent protein of cytoplasmic polyhedrosis virus. At this time, it is important that the open reading frames encoding the constituent protein and the protein of the target protein gene are cloned in-frame. In this way, a recombinant baculovirus expressing the constituent protein of cytoplasmic polyhedrosis virus and the target protein as one fusion protein is constructed, which is described in the above-mentioned invention.
Patent Document 1: International Patent Application WO 02/36785A1
Non-Patent Document 1: Ikeda et al., (2001) J. Virol. 75, 988-995
Applicants herein incorporate by reference the nucleic acid and amino acid sequences of capsid protein VP3 found in FIG. 1 of Non-Patent Document 1 (Ikeda et al., (2001) J. Virol. 75, 988-995).